The broad objective of this proposal is to understand the molecular details of transcription-coupled DNA repair, an important defense mechanism against phenotypic changes induced by insults to DNA in mammalian cells. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of bulky chemical adducts and UV photoproducts in DNA occurs via the same mechanism (excision repair), UV radiation is used as a prototype environmental agent for most of our studies. We will examine the relationship between DNA repair efficiency, transcriptional activity and chromatin structure of two different classes in mammalian genes, and an inducible gene in yeast. The relationship of repair to RNA polymerase II (polII) expression will be examined in mouse cells containing the herpes simplex virus thymidine kinase (tk) gene fused to the mouse mammary tumor virus long terminal repeat (LTR). This "LTL" construction is stably integrated into the genome of these cells and expression of the tk gene requires glucocorticoid hormone. We have shown that hormone-induced transcription of the tk gene is acutely sensitive to UV radiation and returns rapidly following efficient repair in this construct. Since UV photoproducts form preferentially in the LTR region, transcription may be blocked by UV damage to promoter elements. We will use ligation-mediated PCR to examine the yield and repair of UV photoproducts at specific sites in the LTR region required for initiation of pol II. We will also examine the effect of UV damage on the binding of hormone receptor to the LTR using a "gel- shift" assay. Repair of N-methyl purines in ribosomal RNA genes (rDNA) will be examined in mouse Friend erythroleukemia cells. We have fractionated the rDNA of these cells into transcriptionally active and inactive forms of chromatin, using psoralen crosslinking, and find that EcoRI digestion of nuclei releases only the active fraction. Unlike pol II genes, repair of UV photoproducts in both strands of active and inactive rDNA is inefficient and may result from blockage of "bulky" excision repair complexes by the nucleolar compartment. We will determine if inefficient repair of active rDNA also occurs for a different "class" of lesions (N-methyl purines), which are rapidly removed from other genomic sequences by the much smaller base-excision repair proteins. Finally, we are using a simple yeast plasmid, containing an inducible gene and a constitutively expressed gene, as a model chromatin substrate to study transcription-coupled repair in repair proficient (wt) and repair deficient (rad-) yeast cells. We will examine repair at specific sites of this plasmid in "isogenic sets" of wt/rad-cells to determine if specific RAD genes are required for transcription-coupled repair. Thus, we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA lesions. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.